The structure of a conventional biosensor is shown in FIG. 16.
FIG. 16 is a perspective view illustrating the structure of the conventional biosensor test strip.
In FIG. 16, numeral 1 denotes a reactive layer carrier support body composed of a plastic or the like, which supports chromatography materials. Numeral 2 denotes a sample application area composed of a nonwoven fabric or glass fiber filter paper having high absorptivity, or the like, to which an inspection target solution is added or applied, numeral 3 denotes a marker hold region where a marker reagent is held so as to be dissolved, numeral 4 denotes a reactive layer made of nitrocellulose or the like, in which the sample is developed to cause a reaction, numeral 5 denotes a specific protein immobilization part in which a specific protein is immobilized on the region of the reactive later 4, and numeral 6 denotes a water absorbing area which absorbs the inspection target solution finally. The respective regions of the sample application area 2, the marker hold region 3, the reactive layer 4, the specific protein immobilization part 5, and the water absorbing area 6 are formed on the reactive layer carrier support body 1.
An operation of the so-constituted biosensor will be described with reference to FIG. 16.
First, when a liquid sample as an inspection target solution is applied to the sample application area 2, it reaches the area of the marker hold region 3. Then, a marker reagent held in the area of the marker hold region 3 is dissolved due to permeation of the liquid sample and permeates into the region of the reactive layer 4. On the region of the reactive layer 4, there is the specific protein immobilization part 5 in which a specific protein is immobilized, and a reaction is caused between the marker reagent eluted from the area of the marker hold region 3 as well as an analysis target in the liquid sample and the specific protein. At this time, when the analysis target exists in the liquid sample, some color reaction is seen in the region of the specific protein immobilization part 5. The liquid sample is finally absorbed into the water absorbing area 6, and the reaction is ended.
As described above, the biosensor can qualitatively or quantitatively measures components to be measured in a measurement target easily only by the application of the inspection target solution. An example of this biosensor is an immunochromatographic sensor.
A typical immunochromatographic sensor includes an application layer to which the inspection target solution is applied, at least one or plural development layers, and an absorbing layer provided at the end. Further, an antibody immobilization part where an antibody for the measurement target in the inspection target solution is immobilized is provided in a part of the development layer. An antibody for different epitope from the antibody in the antibody immobilization part, which is marked on the upstream side of the antibody immobilization part, is held in a dry state where it can be eluted by the inspection target solution. While mention is made of “different epitope” here, this is not to be regarded as restrictive in a case where an antigen having the identical structure in the identical molecule, such as a dimeric or more antigen, is employed.
Such immunochromatographic sensor has a reaction mode referred to as a sandwich reaction, by which a complex of the antibody immobilized in the antibody immobilization part, the measurement target in the inspection target solution, and the antibody for different epitope from the antibody in the antibody immobilization part is formed.
An operation of the so-constituted immunochromatographic sensor will be described.
First, when a required amount of inspection target solution is applied to the application layer, the inspection target solution permeates into the development layer, and a measurement is started. Then, when the measurement target exists in the inspection target solution, the measurement target can be obtained by the marked antibody bonded to the antibody immobilization part. A typical example of this marked antibody is gold colloid particles, by which visual confirmation is possible, thereby to obtain a measurement result, when the measurement target exists in the antibody immobilization part.
The description has been given here of a case where the sandwich reaction of an antigen antibody reaction, which employs the antibody for the test strip to detect the antigen in the inspection target solution, is taken as a measurement principle. However, the measurement principle is not restricted thereto, and a reaction system in which the antigen is included in the immobilized reagent and the marker reagent to detect the antibody in the inspection target solution may be also employed, and the measurement result can be also obtained by confirming a bonding state of the marker reagent in the antibody immobilization part even when other competitive reactions are similarly taken as measurement principles.
By the way, a biochemical examination of blood is widely implemented as a means for diagnosing the health condition of a person. For example, a measurement of a kind or concentration of a metabolic product, a protein, lipid, an electrolyte, an enzyme, an antigen, an antibody and the like, which are constituents in blood, is performed, while it is hard to perform the measurement directly with whole blood when the above-described immunochromatographic sensor is employed. Generally, to perform the measurement with the whole blood employing the chromatographic sensor, it is required to centrifuge the whole blood first to obtain blood plasma or blood serum as a specimen, and the measurement is performed with the specimen. However, the centrifugation takes labor and time, and thus this chromatographic sensor is unfavorable particularly when a small number of specimens are to be processed immediately or when the examination is to be performed out of doors, at bed side, in the scene of emergency medical care and the like, where facilities for the above operation are not prepared.
In recent years, a quick, simple, accurate, low-cost, and easily available measuring device is desired on the concept of POC (Point of Care) in medical examination scene. The immunochromatographic sensor which can perform a measurement by the application of the inspection target solution is widely utilized for a diagnosis in restricted measurement items and the like as well as in the medical scene owing to its simple measuring operation.
However, according to a biosensor typified by the conventional immunochromatographic sensor, it is difficult to analyze general constituents of blood. That is, for the analysis of the constituents of blood, it is required to centrifuge previously collected blood to obtain blood plasma or blood serum and perform the analysis employing the blood plasma or the blood serum with a large sized analytical instrument. Accordingly, not only a specific machine but also a pretreatment are required for the measurement, so that the examination takes a long time. Therefore, cellular components such as blood corpuscles are affected.
As a method for analyzing constituents of blood with no influence of blood corpuscle components, Japanese Published Patent Applications No. Sho. 57-53661, No. Hei. 8-54387 and No. Hei. 9-196908 disclose a method of filtrating blood corpuscles, by which whole blood is filtrated to separate blood plasma from the whole blood.
For example, according to Japanese Published Patent Applications No. Sho. 57-53661 and No. Hei. 8-54387, to separate blood corpuscle components more completely, glass fiber filter paper with average diameter of 0.2 to 5 μm and density of 0.1 to 0.5 g/cm3 is employed to exude blood, thereby obtaining separated blood plasma and blood serum. However, according to this method, efficiency of blood corpuscle separation is surely enhanced, while it takes quite a long time to almost completely separate the blood corpuscles and a large amount of blood is required to obtain the amount of specimen required for the examination. That is, the amount of blood serum or blood plasma obtained is small with respect to the amount of blood application.
Further, according to Japanese Published Patent Application No. Hei. 9-196908, to prevent clogging in a filtration material due to blood corpuscles and obtain a larger amount of blood plasma or blood serum component from a smaller amount of blood, a water solution of an amino acid or inorganic salt is mixed with whole blood and blood corpuscle components are filtrated thereafter. However, this method requires operations of adding the applying water solution to previously obtained blood and filtrating the blood corpuscle components thereafter, whereby the operation becomes complicated, the measurement takes time, and it is impossible to deal with the examination in an emergency.
To solve the problems, Japanese Patent Application No. 2000-164990 discloses a method of employing a cellular component contraction agent so that cellular components in blood are contracted and develops on a chromatographic test strip. According to this method, the cellular component contraction agent is carried on the test strip so that a blood specimen is applied onto the chromatographic test strip without pretreatment. Although, in this method, the blood specimen can develop on the chromatographic specimen in a short time even without being somehow pretreated previously, a background value is increased and a S/N ratio is decreased due to influence of a blood pigment in the developing blood, whereby a sensitivity in a measurement employing a device is reduced, and the blood pigment prevents reading of a coloration degree, which results in extremely low accuracy for a quantitative measurement.
The present invention is made to solve the above-mentioned problems and has for its object to provide a biosensor which can simply and quickly analyze a measurement target in a liquid sample having a colored component without employing a particular device, and qualitatively or qualitatively analyze components to be measured more accurately.